7282 zyxwvutsrqpon Biochemistry zyxwvu 1989, 28, 7282-7289 Williams, D. H. (1983) Biochemistry 22, 2019-2025. Rinkel, L. J., zyxwvutsrqp & Altona, C. (1987) J. Biomol. Struct. Dyn. Scheek, R. M., Russo, N., Boelens, R., Kaptein, R., & Van Boom, J. H. (1983) J. Am. Chem. SOC. 105, 2914-2916. Scheek, R. M., Boelens, R., Russo, N., Van Boom, J. H., & Kaptein, R. (1984) Biochemistry 23, 1371-1376. Sheth, A., Ravikumar, M., Hosur, R. V., Govil, G., Tan, Z-k., & Miles, H. T. (1987a) Biochem. Biophys. Res. Commun. Sheth, A., Ravikumar, M., Hosur, R. V., Govil, G., Tan, zyxwvuts Z-k., 4, 621-649. 144, 26-34. Roy, K. B., & Miles, H. T. (1987b) Biopolymers 26, Tan, Z-k., Ikuta, S., Huang, T., Dagaiczyk, A., & Itakura, K. (1982) Cold Spring Harbor Symp. Quant. Biol. 47, Widmer, H., & Wuthrich, K. (1987) J. Magn. Reson. 74, Wiithrich, K. (1986) NMR zyxw of Proteins and Nucleic Acids, Wynants, C., & Van Binst, G. (1984) Biopolymers 23, 1301-1 3 13. 383-391. 3 16-336. Wiley, New York. 1799-1 804. NMR Studies of DNA (R+),*(Y-),e(Y+), Triple Helices in Solution: Imino and Amino Proton Markers of TeAoT and C.G.C+ Base-Triple Formation? Carlos de 10s Santos, Mark Rosen, and Dinshaw Patel* Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received April 14, 1989; Revised Manuscript Received May 23, 1989 ABSTRACT: High-resolution exchangeable proton two-dimensional N M R spectra have been recorded on 11-mer DNA triple helices containing one oligopurine (R), and two oligopyrimidine (Y), strands at acidic pH and elevated temperatures. Our two-dimensional nuclear Overhauser effect studies have focused on an 11-mer triplex where the third oligopyrimidine strand is parallel to the oligopurine strand. The observed distance connectivities establish that the third oligopyrimidine strand resides in the major groove with the triplex stabilized through formation of T.A.T and C-GC' base triples. The T-A-T base triple can be monitored by imino protons of the thymidines involved in Watson-Crick (1 3.65-14.25 ppm) and Hoogsteen (12.9-13.55 ppm) pairing, as well as the amino protons of adenosine (7.4-7.7 ppm). The amino protons of the protonated (8.5-10.0 ppm) and unprotonated (6.5-8.3 ppm) cytidines in the C.G.C+ base triple provide distinct markers as do the imino protons of the guanosine (12.6-13.3 ppm) and the protonated cytidine (14.5-16.0 ppm). The upfield chemical shift of the adenosine H8 protons (7.1-7.3 ppm) establishes that the oligopurine strand adopts an A-helical base stacking conformation in the 1 1-mer triplex. These results demonstrate that oligonucleotide triple helices can be readily monitored by N M R at the individual base-triple level with distinct markers differentiating between Watson-Crick and Hoogsteen pairing. Excellent exchangeable proton spectra have also been recorded for (R+),.(Y-),.(Y +), 7-mer triple helices with the shorter length permitting spectra to be recorded at ambient temperature. Our N M R studies open up the possibility of studying triple helices containing errors and lesions, as well as the interaction of ligands with this DNA structural motif in aqueous solution. Eere is currently great interest in the chemistry and biology of triple-stranded DNA helices and their potential role in the regulation of the eukaryotic genome [reviewed in Wells et al. (1988), Htun and Dahlberg (1989) and Broitman and Fresco (1989)l. The early characterization of the poly(U.A.U) triple helix where the third strand was an oligopyrimidine (Felsenfeld et al., 1957) has been complemented by the more recent characterization of the poly(A-U.A) triple helix where the third strand is an oligopurine (Broitman et al., 1987). Related studies established the sequence requirements (Riley et al., 1966; Morgan & Wells, 1968) and the role of pH (Lee et al., 1984) for triple-helix formation. Much of the recent interest in triple-helix formation in- volving zyxwvutsrq (deoxypurine),.(deoxypyrimidine), sequences [desig- nated (R),.(Y),] results from the observation of such tracts 'The research was supported by NIH Grant GM34504. C.d.1.S. is supported, in part, by a fellowship from the CONICET, Republic of Argentina. The NMR spectrometers were purchased from funds donated by the Robert Woods Johnson Trust toward setting up an NMR Center in the Basic Medical Sciences at Columbia University. 0006-2960/89/0428-7282$01.50/0 at recombination hot spots and upstream from eukaryotic genes [reviewed in Wells et al. (1988)]. The detection of S1 nuclease hypersensitivity in active chicken globin chromatin (Larson & Weintraub, 1982) and its fine mapping to (R),.(Y), stretches (Nickol & Felsenfield, 1983; Schon et al., 1983) focused much attention on the unusual DNA structure at such sites. The observed hyperreactivity was explained through (R+),.(Y-),-(Y+), triple-helix formation (the signs indicate the directionality of the strands) with the remaining single- stranded (R-), segment sensitive to single-stranded nucleases (Christophe et al., 1985; Lyamichev et al., 1986). Triple-helix formation in (R),.(Y), mirror repeats is favored by both su- perhelical stress and low pH (Mirkin et al., 1987) and can be readily monitored by two-dimensional gel electrophoresis (Mirkin et al., 1987; Collier et al., 1988) and chemical probes (Hanvey et al., 1988a,b; Voloshin et al., 1988; Htun & Dahlberg, 1988; Johnston, 1988). Finally, chemical foot- printing studies on (G),.(C), sequences in supercoiled plasmid DNA suggest a switch from a (G+),.(C-),.(C+), triple helix in the absence of Mg ions to a (G+),.(C-),.(G-), triple helix 0 1989 American Chemical Society